The Roles Of Salicylic Acid Metabolism Of Metarhizium Acridum In Plant Disease Resistance

Metarhizium acridum,an entomopathogenic fungus,plays a pivotal role in regulating the population of harmful insects.Salicylic acid（SA）,a phenolic compound that can activate the immune response of plants to pathogens.Our previous work showed that the salicylate synthase gene MaSalS existed in M.acridum that can produce salicylic acid and secreted outside the cell.In this study,the effects of M.acridum on plant disease resistance were studied using the wild type of M.acridum（WT）,Arabidopsis thaliana（Col-0）and Pst DC3000 as materials.TheΔMaSalS mutant was used to study the effect of salicylate metabolism in M.acridum on plant disease resistance.Through transcriptome sequencing（RNA-seq）,the mechanism of M.acridum and its salicylate metabolism in affecting plant disease resistance were studied.Results of this study are helpful elucidating the mechanism of interactions between fungal insecticides and plants,our work also provide the reference for application of entomopathogenic fungi to control plant diseases.At the same time,it also lays a theoretical foundation for the breeding and application of fungal insecticides with high efficiency,and provides new clues for the study on the interaction between other fungi and plants.The main results of this study are as follows:（1）M.acridum did not affect the growth of Pst DC3000 in vitro.M.acridum spores did not germinate on the leaves of Arabidopsis thaliana and without infection.（2）The application conditions of Ma102 and Pst DC3000 were optimized.Gradient concentration of WT conidia suspension was sprayed on the seedlings.q RT-PCR was performed to analyze the transcription level of pathogen related gene PR1.we found that the optimal spraying concentration of the conidial suspension was5×10⁸ spores/m L/pot（5 seedlings each pot）.Arabidopsis thaliana was treated with Pst DC3000 of different concentrations.The optimal volume of Pst DC3000 was 1 m L/pot with concentration of OD₆₀₀=0.05 with Optimal pretreatment time of Ma102 was co-treated with Ma102 and Pst DC3000,which the seedlings showed the strongest disease resistance.（3）Plant disease resistance could be induced by M.acridum.After treated with M.acridum,infection symptoms and disease index of A.thaliana decreased,CP treatment had the same effect with WT group.Meanwhile,the pathogen-related gene PR1 was dramatically induced by M.acridum and population of Pst DC3000 in the plant leaves drastically decreased.The H₂O₂ content in A.thaliana was also boosted up by the treatment of Ma102.All of these evidences suggested that the resistance to Pst DC3000in A.thaliana was strengthened by Ma102.（4）Plant disease resistance could be improved by endogenous SA of M.acridum.The resistance to Pst DC3000 in A.thaliana also could be enhanced by the treatment withΔMaSalS,while moderated effect was observed compared to WT.These results suggested that disruption of the MaSalS gene dramatically reduced the improvement of disease resistance to Pst DC3000 of M.acridum in A.thaliana.（5）The expression of genes related to plant immune signaling could be induced by M.acridum.after co-treated with WT and Pst DC3000,1027 genes were found differentially expressed at 6 h by RNA-seq compared to control.DEGs mainly enriched in the GO terms of secondary metabolite biosynthetic process（54）,response to bacterium（60）,immune system process（50）,response to SA（29）,glutathione transferase activity（14）and oxidordeuctase activity（37）.some DEGs were involved in biosynthesis of plant antagonistic substance and plant immune signaling,including glucosinolate biosynthesis（12）,phenylpropanoid biosynthesis（15）,glutathione metabolism（13）and MAPK signaling pathway（13）.These results demonstrated that pretreatment with M.acridum could improve the resistance to Pst DC3000 in A.thaliana.After 12 h treatment with Ma102,157 DEGs were obtained,in which genes involved in plant immunity were reduced compared to 6 h treatment results.There were only 11 and 9 DEGs related to bacterial response and immune response process respectively.（6）Induction of genes involved in plant immunity were decreased by disruption of the MaSalS gene in M.acridum.After 6 h treatment,according to RNA-seq analysis,only 211 DEGs were detected by treated withΔMaSalS,which 1179 DEGs were reduced compared with WT treatment.Accordingly,DEGs enriched in the same GO terms decreased.There were 70 DEGs in terms of bacterial response,59 DEGs in terms of immunity process,and 37 DEGs in terms of response to SA.Moreover,DEGs in WT treatment enriched in 11 KEGG pathways related to plant disease resistance,while only4 KEGG pathways enriched inΔMaSalS treatment,and the amount of DEGs was also dramatically reduced in the same pathway.For instance,10 DEGs reduced in glucosinolate biosynthetic process.In conclusion,in this study,the effect of M.acridum and its SA metabolism on the disease resistance to A.thaliana was analyzed in a physiological and molecular level.the molecular mechanism of plant disease resistance induced by M.acridum and its salicylate metabolism by RNA-seq.Bioinformatics analysis showed that other entomogenic fungi that had been sequenced also had homologous genes of MaSalS,so they might have similar salicylic acid synthesis pathway.Therefore,insect pathogenic fungi may generally activate plant disease resistance.